SITE-DIRECTED MUTAGENESIS AND SUGAR-BINDING PROPERTIES OF THE WHEAT-GERM-AGGLUTININ MUTANTS TYR73PHE AND PHE116TYR

Wheat germ agglutinin is a dimeric lectin composed of two identical su bunits. Each subunit consists of four homologous hevein-like domains o f 42 or 43 amino acids each. Amino acid residues at the same position in each domain involved in sugar binding are thought to play a similar role in sugar binding. In order to clarify the role of the amino acid residue at domain position 30 of wheat germ agglutinin isolectin 2 (W GA2) in sugar binding, two WGA2 variants each containing a mutation, e ither Tyr73-->Phe (domain B) or Phe116-->Tyr (domain C), were produced . The binding activity for (GlcNAc)(3) and the three-dimensional struc ture of these mutants were characterized by comparing with the propert ies of wild-type WGA2. Equilibrium dialysis experiments using (GlcNAc) (3) indicated that the mutation Tyr73-->Phe reduced the overall sugar- binding activity at both pH 5.9 and pH 4.7. In addition, positive coop erativity toward (GlcNAc), binding was observed at pH 4.7. In contrast , the mutation of Phe116-->Tyr increased the overall sugar-binding act ivity at pH 5.9, but reduced this activity at pH 4.7 without changing the number of sugar-binding sites. Positive cooperativity was not obse rved at pH 5.9 or pH 4.7. X-ray crystallographic analysis of mutant WG A2 revealed that the mutation of Tyr73-->Phe caused a side chain movem ent of the Glu115 residue of the opposite subunit that formed a hydrog en bond with Tyr73 in wild-type WGA2. No changes were observed in the backbone structure and the disposition of the benzene ring of Phe73. T he mutation Phe116-->Tyr caused the formation of a new hydrogen bond b etween Tyr116 and Glu72 of the opposite subunit. The changes in the su gar-binding properties in WGA2 mutants are discussed in relation to th e structural change at the binding site.